WORLD CLASS BASIC, TRANSLATIONAL AND CLINICAL RESEARCH

Overview

The main focus of the Cell Signalling group is the identification of therapeutic targets in lung cancer, the most common cause of cancer related deaths worldwide. Lung cancer is divided into non-small cell lung cancer (NSCLC, approximately 85% of cases) and SCLC (~15% of cases). The most common histological subtype of NSCLC is adenocarcinoma, of which the most common driver mutation is KRAS. KRAS mutant lung adenocarcinoma (KRASm-LUAD) and SCLC treatments lag behind other lung cancer types, for which targeted therapies offer additional treatments prolonging patient survival. No approved targeted therapies exist for SCLC, and until very recently, nor did they for KRASm-LUAD. However, recently direct inhibitors of KRASG12C (accounting for ~40% of KRASm-LUAD cases) have been approved, but resistance has been documented in relapsing patients. Other KRASm isoforms lack effective targeted therapies. Therefore, current drug development efforts focus not only on KRAS itself, but also on downstream targets. One such downstream target under investigation in our laboratory is the small GTPase RAC1.

RAC is a member of the RHO-like family of GTPases and cycles between a GDP- and a GTP-bound state. When GTP-bound, it interacts with various effector molecules that regulate several cellular processes, including proliferation and migration. Multiple mechanisms control RAC activity, including control of nucleotide binding and hydrolysis by guanine nucleotide exchange factors (GEFs) and GTPase Activating Proteins (GAPs) respectively, regulation of subcellular localisation and modulation of RAC protein levels (reviewed in Porter et al., Small GTPases 2017). Moreover, several studies using recombinant RAC and RAC GEF mice have shown that RAC is required for the formation and growth of tumours. In particular, RAC is required for the formation of KRAS-induced lung tumours in mice. Moreover, the RAC GEF TIAM1 is required for the formation and growth of HRAS-induced skin tumours (Malliri et al., Nature 2002). Interestingly, TIAM1 and its homologue STEF/TIAM2 both contain a RAS-binding domain and are considered RAS effectors.

Role of TIAM-RAC1 signalling in lung cancer formation and progression

The role of TIAM1 and STEF/TIAM2 in NSCLC formation and progression is currently under investigation using in vitro and in vivo models and updates will be given in subsequent annual reports. SCLC is a highly aggressive malignancy broadly divided into neuroendocrine (NE, >80% of SCLC) and non-NE subtypes. Analysis of expression data from SCLC tumours, patient-derived models and established cell lines, showed that the expression of TIAM1 is associated with a neuroendocrine gene program (STEF/TIAM2 levels were very low in all SCLC tumours/cell lines). Moreover, we showed that TIAM1 depletion or RAC1 inhibition reduces viability and tumorigenicity of SCLC cells by increasing apoptosis associated with conversion of BCL2 from its pro-survival to pro-apoptotic function via BH3 domain exposure. We showed that this conversion is dependent upon cytoplasmic translocation of Nur77, an orphan nuclear receptor. Like in other cell types, TIAM1 is present in the nucleus of SCLC cells, where it interacts with Nur77 sequestering it in SCLC cell nuclei. TIAM1 depletion or RAC1 inhibition promoted Nur77 translocation to the cytoplasm. Importantly, mutant TIAM1 with reduced Nur77 binding failed to suppress apoptosis triggered by TIAM1 depletion. In conclusion, TIAM1-RAC1 signalling promotes SCLC cell survival via Nur77 nuclear sequestration (Payapilly et al (2021). TIAM1-RAC1 promote small-cell lung cancer cell survival through antagonizing Nur77-induced BCL2 conformational change. Cell Reports, 37(6), 109979.).

Role of RAC1 activators in migration and malignant progression

Although RAC seems always to promote tumour formation and growth, it may promote or antagonise malignant progression. There are cases where deletion of RAC GEFs leads to more invasive tumours and there are reports suggesting that reduced RAC activity levels correlate with more aggressive tumours (Porter et al., Small GTPases 2017). In vitro data have shown that activation of RAC may lead to opposing migratory phenotypes raising the possibility that targeting RAC in a clinical setting could exacerbate tumour progression. For these reasons it is important to identify the factors that influence whether RAC activation will promote or inhibit migration. One such factor that we have identified is the GEFs that activate RAC. RAC GEFs are multi-domain proteins with many binding partners. We showed that TIAM1 and another RAC GEF, P-REX1, have diametrically opposite effects on cell migration through RAC in certain epithelial cells and fibroblasts: TIAM1 promotes cell-cell adhesions to oppose cell migration while P-REX1 promotes migration. They perform these contrasting roles in cell migration by selecting RAC effectors (Marei et al., Nat Commun 2016). Over-expression of specific GEFs, which occurs commonly in many cancers, can therefore drive different oncogenic signalling pathways. These data on TIAM1 are consistent with the fact that even though TIAM1 knockout mice were resistant to the formation of RAS-induced tumours, the few tumours which did form were more aggressive (Malliri et al., Nature 2002). This highlights two distinct roles for TIAM1/RAC signalling: stimulating tumour formation but suppressing malignant progression. Early work on TIAM1’s role in suppressing migration and invasion focused on its role in strengthening cell-cell junctions, associated with anti-migratory effects (Malliri et al., J Biol Chem 2004). It was also shown that cell-cell adhesion disassembly and scattering of epithelial cells requires depletion of TIAM1 from cell-cell adhesions (Woodcock et al., Mol Cell 2009; Vaughn et al. Cell Rep 2015).

But besides these studies showing that TIAM1 inhibits migration by promoting cell-cell adhesion, we have also identified another mechanism by which TIAM1 hinders migration. TIAM1 localises in the nucleus of several colorectal cancer cell lines and nuclear TIAM1 inhibits their migration via suppressing the interaction of the transcriptional co-activator TAZ with its cognate transcription factor TEAD. Suppression of this interaction by TIAM1 inhibited expression of TAZ/YAP target genes implicated in epithelial-mesenchymal transition and cell migration. Consistent with these in vitro data, we showed by staining a microarray of colorectal cancer biopsies that TIAM1 localised to the nuclei of tumour cells. Moreover, nuclear staining intensity significantly decreased with advancing Dukes stage and patients with high nuclear TIAM1 had significantly better survival than those with low nuclear TIAM1 (Diamantopoulou et al., Cancer Cell 2017).

More recently, we have uncovered a new role for TIAM1 in regulating the duplication of centrioles – structures at the core of centrosomes found at the poles of the mitotic spindle. Cells normally duplicate centrioles only once per cell cycle. Centriole overduplication is common in many cancers however, promoting aneuploidy but also increasing invasiveness of tumour cells. We found that TIAM1 localises to centrosomes and that its depletion leads to centriole overduplication, lagging chromosomes at anaphase and aneuploidy (Porter et al., J Cell Sci. 2021), indicating another mechanism by which loss of TIAM1 could promote malignant progression.

Apart from these roles of RAC1 activators in antagonising cell migration, RAC1 activators are also associated with promotion of cell migration. Interestingly, activation of RAC by STEF/TIAM2 promotes migration (Rooney et al., EMBO Rep. 2010), something we have seen in all cell types tested. STEF/TIAM2 localises at the nuclear envelope, co-localising with the key perinuclear proteins Nesprin-2G and Non-muscle myosin IIB, where it regulates perinuclear RAC1 activity. Interestingly, STEF depletion reduced apical perinuclear actin cables (thick actin bundles which run over the nucleus, constraining its height and guiding nuclear orientation), increased nuclear height and impaired nuclear re-orientation, which is required for optimal cell migration. Finally, STEF depletion reduced expression of TAZ-regulated genes, indicating an alteration in mechanosensing pathways as a consequence of disruption of the actin cap.